Severe Thrombocytopenia and Platelet Granule Dysfunction Associated with the Combination of Novel Heterozygous Variants in TUBB1 and ITGB3

Scientific Research Question: The TUBB1 gene encodes tubulin β-1 chain, which is the predominant β-tubulin isotype expressed in megakaryocytes and platelets. Tubulin β-1 chain is involved in microtubule assembly and assumed to be involved in proplatelet formation and platelet release. Microtubules are presented as marginal band in resting platelets providing the structural integrity. Autosomal dominant macrothrombocytopenia has been shown to be related to TUBB1 or ITGB3 (encoding the β 3 subunit of the integrin aIIbβ3) variants, which is each accompanied by mild-moderate thrombocytopenia and heterogeneous bleeding severity. Here, we report a 4-year-old male patient with very frequent haematoma, petechiae, easy bruising since birth, medical treatment of bleeding from minor wounds (ISTH-BAT score: 6) and severe thrombocytopenia (22,000/µl), MPV 8.3 fl, despite normal leukocyte count and haemoglobin/haematocrit. Platelet-type von Willebrand disease, von Willebrand disease type IIb, Wiskott-Aldrich-syndrome, autoimmune and MYH9-related thrombocytopenia were excluded. His non-consanguineous parents had no bleeding diathesis and normal platelet counts.

Methodology: Platelet phenotype was analyzed by flow cytometry in citrated-whole blood (Jurk K. Hämostaseologie 2015) and by immunofluorescence labeling of standard air-dried peripheral blood smears (Greinacher A et al. JTH 2017). Platelet function analysis was performed in platelet-rich plasma using specific flow cytometric assays. Whole exome and Sanger sequencing was used for genetic analyses.

Findings: The majority of platelets were normally sized and only a few large platelets were detected in the patient's blood smear. The patient's platelets expressed up to 50% decreased integrin aIIbβ3 surface levels, whereas other major platelet receptor surface levels were in the normal range. ADP- and TRAP-6-induced platelet PAC-1-binding was impaired. Immunofluorescence labeling of platelet granule proteins revealed normal counts and distribution of a-, d-granules and lysosomes. However, thrombin- or convulxin-induced exocytosis of all three granule types was severely impaired. Immunofluorescence labeling of platelet tubulin β-1 chain demonstrated disturbed marginal microtubule bands. Whole exome sequencing identified previously undescribed heterozygous variants in TUBB1 (c.862G>A predicting a p.E288K substitution) and in ITGB3 (c.2221T>C predicting a p.W741R substitution). Interestingly, the mother carried the same heterozygous variant in TUBB1, whereas neither the mother nor the father carried the heterozygous variant in ITGB3.

Conclusion: We describe for the first time that the combination of novel heterozygous variants in TUBB1 and ITGB3 leads to severe thrombocytopenia and platelet dysfunction characterized by defects of global granule secretion and of the integrin aIIbβ3.